Project summary The pro-inflammatory polarization of monocytes/macrophages is a hallmark of low-grade inflammatory syndromes such as impaired wound healing. Despite its significance, the molecular mechanisms controlling monocyte low-grade inflammatory polarization is not clearly understood. This presents a major impediment for the development of effective treatments for chronic diseases associated with low-grade inflammation. Bacterial endotoxin Lipopolysaccharide (LPS) is a powerful agent mediating monocyte polarization, causing complex and dynamic alterations in monocyte activation, priming and tolerance in a concentration-dependent fashion. One of the least studied, yet highly important effect of LPS is manifested in the sustained low-grade inflammatory polarization by subclinical super-low dose LPS. Low-grade circulating endotoxemia is prevalent in humans with adverse conditions such as obesity and diabetes. We demonstrated that subclinical super-low dose LPS preferentially polarizes monocytes into a low-grade pro-inflammatory state with elevated IRF5 levels. In vivo injection of super-low dose LPS programs innate immune environment into a low-grade inflammatory state and delays wound repair. Mechanistically, we observed that super-low dose LPS generates cellular stress, halts the homeostatic process of lysosome fusion, and stabilizes IRF5. We identified a novel innate signaling molecule Tollip that is critically important for maintaining innate leukocyte homeostasis through maintaining proper lysosome function. Super-low dose LPS clears away Tollip from lysosome, disrupts lysosome homeostasis and autophagy completion, and contributes to the stabilization of the key low-grade inflammatory monocyte signature transcription factor IRF5. Complementing our novel mechanistic studies, we obtained in vivo data that show reduced wound repair and increased inflammation in wild type mice injected with super-low dose LPS, as well as in Tollip deficient mice. A compound derived from bile acid, TUDCA, can potently retain Tollip within lysosome and restore lysosome function. We observed that the application of TUDCA can hasten wound healing. Our long-term goal is to define novel therapeutic targets for maintaining a proper balance of immune environment and treating chronic wounds associated with low-grade inflammation. Based on our novel observations, our objective is to define molecular mechanisms by which super-low dose LPS programs innate monocytes into a low-grade pro-inflammatory state and impairs proper wound healing. Our central hypothesis is that super-low dose LPS polarizes a low-grade pro-inflammatory monocyte state and delays wound healing through the disruption of cellular homeostasis maintained by Tollip. To test this hypothesis, we plan to perform the following integrated studies. Aim 1 will test that the disruption of lysosome function is critical for the IRF5 stabilization and inflammatory polarization of monocyte. Aim 2 will examine the clearance mechanism of lysosomal Tollip in monocytes during the low-grade inflammatory polarization by super-low dose LPS. Aim 3 will test the hypothesis that monocyte polarization plays a key role in compromised wound healing. Completion of this project will define novel mechanisms responsible for the pro-inflammatory polarization of monocytes, and facilitate the development of therapeutic strategies in treating chronic wounds associated with low-grade inflammation.